A. Field of the Invention
The present invention is related to a process that noticeably improves the bioavailability of carotenoids by obtaining carotenoid micelles in a lipid matrix in the presence of water. Such lipid matrix is composed of the free fatty acids to which the xanthophylls are naturally bound, and the waxes, phospholipids and sterols that naturally occur in the carotenoids' extracts, as well as emulsifying agents. During the esterification reaction of lutein, zeaxanthin, and other carotenoids with short chain organic acids, such as acetic or propionic acids, and further processing, the formation of carotenoid micelles occur in a lipid matrix that have been found to be readily absorbed through the intestinal wall. Such absorption is a noticeable improvement as compared to the lower bioavailability of carotenoid crystals. The invention also relates to formulations of carotenoid microemulsions and nanoemulsions that improve the bioavailability in humans, poultry and marine organisms.
B. Description of the Related Art
Carotenoids are terpenoid compounds that besides their typical pigmenting characteristics (yellow, orange or red pigments), function as precursors of molecules with biological activity intervening in different vital biological and physiological processes.
Over 600 different carotenoids have been recognized in nature. Carotenoids are classified in two major groups: carotenes, which are hydrocarbon molecules comprising atoms of carbon and hydrogen only. Representative examples of carotenes include β-Carotene and Lycopene. And xanthophylls, which are oxygenated derivatives of the carotenes. Examples of xanthophylls include Lutein, Zeaxanthin, Isozeaxanthin, Capsanthin, Capsorubin, Cryptoxanthin, Astaxanthin, 3′ Epilutein, and Cantaxanthin.
Carotenoids are widely distributed in nature. Total annual production in nature is estimated at over 100 million tons. Carotenoids intervene in the physiology of all living organisms. They are produced in nature by photosynthetic and enzymatic reactions carried by marine microorganisms as microalgae, bacteria, fungii and zooplancton; and in most terrestrial living plants occur in their leaves, flowers and fruits.
In flowers and fruits, carotenoids impart vivid yellow, orange and red colors.
In birds, carotenoids play the role of vital functions as well as cosmetic purposes, they differentiate genders, and are indicative of sexual maturity and attraction.
In marine organisms, carotenoids are more abundant than in terrestrial organisms and they are responsible for several vital biological, physiological, metabolic and reproductive functions. Carotenoids provide color to marine microalgae and bacteria, krill, salmon, trout, red sea bream, yellow tail tuna, crustaceans, etc.
No animal species are capable of synthesizing carotenoids. Therefore, they must obtain their requirements through their diet. Broilers and layers grown in captivity require a given dose of lutein and zeaxanthin in their feed in order to supplement their requirements. Laying hens accumulate lutein, zeaxanthin and cantaxanthin in the yolk, protecting the embryo against the oxidative damage provoked by the free radicals that occur due to the high rate of metabolic reactions in the rapidly developing embryo. Broilers, on the other hand, accumulate lutein and zeaxanthin in their adipose tissue as well as in the skin. Such deposits act as reservoirs of carotenoids, and become available when required to perform specific physiological functions.
Some carotenoids are metabolized by terrestrial or marine organisms into Vitamin A, and such carotenoids are the only source of Vitamin A for herbivores or omnivores living in their natural environment.
Carotenoids act as effective antioxidants in most living organisms. They have the capability to quench free radicals that are produced in metabolic reactions at the cellular level, avoiding tissue degradation.
Extensive research in the past years indicate that the presence of lutein and zeaxanthin in the macula helps the prevention of age related macular degeneration in humans, as well as avoiding the development of cataracts. Lutein and zeaxanthin are also present in human breast milk, and in the adipose tissues.
Dark green leafy vegetables, tomatoes, as well as yellow corn, and many fruits like oranges, mangoes, grapefruit, etc. are the natural source of dietary carotenoids. Carotenoids occur in a concentrated way in chromoplasts attached to proteins or fibers by non-covalent links. However, chloroplast or chromoplast and other plant structural materials may not be the ideal source of carotenoids for human consumption due to their low bioavailability. Carotenoids occur in food plants as part of the photosynthetic apparatus (green leafy vegetables), dissolved in oil droplets (fruits) or as semi crystalline membrane-bound solids (carrot, tomato). Fatty acid esters of carotenoids, such as lutein, zeaxanthin and cryptoxanthin, occur in some fruits (peaches, papaya, peppers), as well as in xanthophyll concentrates obtained from marigold (Tagetes erecta) and red peppers (Capsicum annum).
To insure that the carotenoids are absorbed by the organism, they should first be freed from their chromoplast matrix. They are thought to be hydrolyzed in the intestinal lumen before mucosal uptake, most likely by the carboxylic ester hydrolase secreted by the pancreas.
In animals, the absorption of carotenoids is generally accepted as following the absorption of lipids—i.e. emulsification and incorporation into mixed micelles, which are then absorbed by the mucosa of the small intestine, mainly in the duodenum, in parallel with fat digestion and absorption. They are transported through the unstirred water layer and taken up by the enterocytes by passive diffusion.
It is in the intestinal mucous wall that such passive diffusion is improved if the carotenoids are dissolved in a lipid forming a micelle in an aqueous media, rather than if they are in crystalline form. Crystals are not the ideal physical form for this diffusion to occur, nor are carotenoids crystals readily incorporated into micelles. If the carotenoids enter the gut already in the form of microemulsions, or as nanoemulsions, the formation of micelles is facilitated.
After the carotenoids are absorbed, they are transported through the enterocytes from the luminal side to the serosal side. They are packaged in chylomicrons and secreted into the thoracic duct, and find their way into the circulating blood via the vena cava inferior.
Among the main factors that affect the bioavailability of xanthophylls by organisms are: physical form in the source (food matrix), the structure of the xanthophylls molecules, and the interaction of the xanthophylls with other nutrients, mainly lipids. Therefore, it would be highly desirable to develop carotenoid compounds, derivatives or formulations with improved stability, absorption and bioavailability.
Many natural and processed foods consist either partly or wholly as emulsions or have been in an emulsified state at some time during their production. The manufacture of an emulsion-based food product with quality attributes depends on the selection of the most appropriate raw materials (e.g., water, oil, emulsifier, thickening agents, minerals, acids, colorants, flavours, vitamins, etc.) and processing conditions (e.g.—mixing, homogenization, pasteurizations, sterilization, etc.).
An emulsion is a mixture of two immiscible liquids (usually oil and water), with one of them dispersed as small spherical droplets in the other phase. Emulsions can be conveniently classified according to the distribution of the oil and aqueous phases. A system which consist of oil droplets dispersed in an aqueous phase is called an oil-in-water or O/W emulsion (e.g., mayonnaise, milk, cream, soups and sauces). A system which consists of water droplets dispersed in an oil phase is called a water-in oil or W/O (e.g., margarine, butter and spreads). Emulsions are part of a more general class of two-phase systems of matter called colloids. Although the terms colloid and emulsion are sometimes used interchangeably, emulsion tends to imply that both the dispersed and the continuous phase are liquid. An emulsifier (also known as a surfactant from surface active material or emulgent) is a substance which stabilizes an emulsion.
The main role of the surfactants in food emulsions is to enhance their formation and stability. Surfactants used in the food industry are mainly nonionic (e.g., monoacylglycerols, sucrose esters of fatty acids), anionic (e.g., fatty acids), or zwitterionic (e.g., lecithin). Surfactants aggregate spontaneously in solution to form a variety of thermodynamically stable structures know as association colloids (e.g., micelles, bilayers, vesicles, and reverse micelles). The shape of a micelle is controlled largely by the molecular geometry of its surfactant molecules, but micelle shape also depends on the conditions, such as temperature or pH, and the type and concentration of any added salt.
Nonpolar molecules, which are normally insoluble or only sparingly soluble in water, can be solubilized in an aqueous surfactant solution by incorporation into micelles or other types of association colloids. Micelles containing solubilizied materials are referred to as microemulsions or swollen micelles, whereas the materials solubilizied within the micelle are referred to as the solubilizate.
In view of the above referred needs, applicants developed a process for obtaining a microscopic physical state of xanthophylls or oxycarotenoids derivatives forms, such as diacetates and dipropionates derivatives, that are readily incorporated in the digestive system as micelles providing an improved bioavailabilty, as compared to the bioavailability of carotenoids that are ingested in crystalline form.
U.S. Pat. No. 2,861,891 issued to Bauernfeind and Howard describes a process to obtain a dry powder, obtaining a supersaturated carotene solution by heating in a vegetable oil that afterwards is dispersed in an aqueous gelable colloid solution, and converting the emulsion thus formed into a dry particulate form. Such process involves heating the carotenoids in vegetable oil in order to improve the solubility of the carotenes, lycopene, lutein, zeaxanthin, cryptoxanthin, bixin and methyl bixin, and avoiding the precipitation of carotenoids crystals by incorporating a gelable colloid. A further emulsification step follows in an aqueous colloidal solution that forms a gel capable of producing a dry powder after a spray drying process. The product obtained is a microcrystalline dispersion in edible oil used to impart color to margarine, fruits and vegetables in which the βcarotene occurs in the form of microdispersion in a protective hydrophilic colloid.
U.S. Pat. No. 3,523,138 issued to Grant describes the improved bioavailability of carotenoids in poultry, after a saponification reaction has taken place in order to free the xanthophylls from the long chain fatty acid esters.
U.S. Pat. No. 3,535,426 issued to Hawks discloses that a saponified mixture of xanthophylls become more stable, and consequently more bioavailable, when admixed with a fat and ethoxyquinolein.
Cathrein describes in U.S. Pat. No. 5,364,563 a process for producing powdered carotenoid preparations by obtaining a suspension in oil that is brought in contact with superheated steam, producing an emulsion that is spray dried.
Eugster et al, in U.S. Pat. Nos. 5,496,813 and 5,536,504 obtain ultra microemulsions that form spontaneously dispersible concentrates containing xanthophylls esters that have anti tumor activity.
Gellenbeck in U.S. Pat. No. 5,827,539 obtains a finely ground mixture of carotenoids with an oil and a spray dried encapsulated form that is water dispersible.
Luddecke, et al, in U.S. Pat. No. 5,863,953 describes the obtention of an oil dispersion of carotenoids, which is used to prepare a double dispersion system with particles sizes of 100 microns, stabilized by a protective colloid and emulsifiers.
Sanders and Herink published in WO9947001 the increased bioavailability of lutein and zeaxanthin in humans and poultry using isolecithin and lecithin.
Kolter et al, describe in U.S. Pat. No. 5,891,907 stable aqueous solubilizates of carotenoids and vitamins, in which the carotenoids and the water insoluble vitamins with the aid of a nonionic emulsifier, yield a micelle, which particles are smaller than 100 nm.
Luddecke et al, describe in U.S. Pat. No. 5,895,659 the preparation of finely dispersed carotenoids or retinoid suspensions by dissolving the carotenoid or retinoid in a volatile, water-miscible organic solvent, under elevated pressures, and immediately after 10 seconds, mix the solution with an aqueous medium containing an emulsifier.
Schweikert et al in U.S. Pat. No. 5,925,684 describe a stable oil in water emulsion consisting of an aqueous phase and an oil phase which is very finely dispersed by means of an emulsifier, and wherein the carotenoid is present in the oil phase in a concentration above the saturation solubility of the carotenoid in the oil at room temperature.
Auweter et al, describe in U.S. Pat. No. 5,968,251 the preparation of coldwater dispersible powders by preparing a molecular-disperse solution of a carotenoid, with or without an emulsifier and/or edible oil, in a volatile, water-miscible organic solvent at elevated temperature and adding an aqueous solution of a protective colloid; whereupon the hydrophilic solvent is transferred into the aqueous phase, and the hydrophobic phase of the carotenoid results as a nanodisperse phase, removing the solvent by heating the hydrosol and converting it in a water dispersible dry powder.
Gellenbeck in U.S. Pat. No. 5,976,575 describes the grinding of a mixture of carotenoids and oil to reduce the carotenoids particle size, emulsifying the mixture with an encapsulating mixture and drying the emulsion.
Handelman in U.S. Pat. No. 6,075,058 describes the composition of lutein and zeaxanthin along with cholesterol, olive oil, egg yolk phospholipids, alpha tocopherol and aqueous sodium chloride. The mixture is prepared by mixing the lipid ingredients into ethanol, evaporating the ethanol, and dispersing the lipids as an emulsion in the sodium chloride solution.
Kowalski et al, describe in U.S. Pat. No. 6,093,348 a method for the manufacture of carotenoid powders, weherein an aqueous suspension of the carotenoid is heated to melt the cardtenoid in the presence of a surfactant and a protective colloid under high temperatures and high pressures (HTHP process). The suspension is then homogenized under high pressure to form an emulsion. And the resulting emulsion is dried to obtain the carotenoid powder.
Schliapalius in U.S. Pat. No. 6,132,790 describes a composition of a carotenoid in oil, a dispersion of a water dispersible matrix and a stabilizer, and a non-oil solvent and an emulsifier, all of natural sources.
Koguchi, et al describe in U.S. Pat. No. 6,261,622 a method to provide a water-dispersible carotenoid preparation which can be added to various aqueous compositions with retaining dispersion stability even at low temperature, by dispersing pulverized carotenoids crystals with soybean extract fibers as emulsion stabilizer.
Bewert et al, in U.S. Pat. No. 6,328,995 describe a procedure to stabilize dry powders which are insoluble in hot water and which contain one or more lipid soluble vitamins or carotenoids, formed in an aqueous dispersion containing a protein, a sugar and potassium and/or sodium phosphates.
Stein et al, describe in U.S. Pat. No. 6,406,735 a process for the preparation of a pulverous composition of a finely divided carotenoid or retinoid comprising; forming a suspension of the active ingredient in a water-immiscible organic solvent containing an antioxidant and/or an oil, feeding the suspension through a heat exchanger and heating the suspension to a high temperature for a 5 seconds residence time, rapidly mixing the solution with an aqueous swellable colloid and further removing the organic solvent to obtain the pulverous preparation, all steps processed in a continuous sequence.
Guerra-Santos, et al describe in U.S. Pat. No. 6,936,279 the obtention of microcrystalline form of carotenoids, particularly zeaxanthin, in an oily carrier. The “coarse-grained” carotenoids is dissolved in a suitable solvent as tetrahydrofuran, and mixed with a vegetable oil and an emulsifier. The mixture is injected along with an inert gas into a vacuum chamber in order to remove the solvent in a flash manner, not allowing the carotenoids crystals to grow. They obtain a microcrystalline suspension in the oil carrier.
All of the above references are related to carotenes, carotenoids or oxycarotenoids in their free and pure form and in no instance refer to carotenoids or oxycarotenoids derivatives, such as diacetates, or dipropionates or to any carotenoids or oxycarotenoid compound or derivative alone or as a complex.
Therefore, to the best of our knowledge, the process and formulation disclosed herein has not been disclosed in the prior art. Furthermore, none of the above described patents completely avoids the formation of carotenoids crystals, which as previously described, affect the absorption and bioavailability of carotenoids.
By the process of the present invention, it is possible to obtain microemulsions or nanoemulsions of lutein, 3′epilutein, and zeaxanthin diacetates and dipropionates, as well as short chain diesters of capsanthin, capsorubin, astaxantin and the acetate and propionate of cryptoxanthin that readily form micelles that are absorbed in the gut and diffuse through the mucous intestine wall.
Crystalline solids are composed of atoms, ions, or molecules in a highly ordered geometric pattern referred to as the crystal lattice. The atoms, ions or molecules are held in their positions by electrostatic, dipole and/or London forces. When a pure crystalline solid is heated, the atoms, ions or molecules vibrate more and more rapidly until at a definite temperature the thermal motion of the particles becomes great enough to overcome the forces of attraction. Then the atoms, ions or molecules enter a more random and mobile state, the liquid state. The melting point of a solid is defined as the temperature at which the liquid and solid phases are in equilibrium. A pure solid will generally melt sharply because the forces of attraction between the particles are the same. However, the presence of a foreign particle in a crystal lattice interrupts its uniform structure and the forces of attraction are weakened. An “impure” compound melts at a lower temperature and over a wider range. Thus, in the process of the present invention, the melting point of carotenoid derivatives is lowered (depressed) by the addition of a soluble material to the solution.
It is a common practice in the pigment industry to carry on a saponification reaction, or hydrolysis, of the fatty acids diesters of lutein and zeaxanthin as they naturally occur in the oleoresin of Tagetes erecta, in order to free the above mentioned carotenoids. It is also a common practice to hydrolyze the oleoresin of Capsicum annum to free the capsanthin and capsorubin from the fatty acids, as they occur in their natural form.
Such hydrolysis is carried out either in an aqueous media by means of a strong alkali and suitable emulsifiers and temperature, or in a non-polar organic media, such as propylene glycol, also under the action of a strong alkali and temperature. In both cases, as the xanthophylls are free they become insoluble in the reaction media and occur in crystalline form.
The reaction mass can be used to prepare pigment premixes or water dispersions, but the physical structure of the carotenoids is always microscopic crystals.
In the different purification and refinement processes aimed to obtain carotenoids of high purity, the saponified oleoresin mass is subject to several stages of selective organic polar, or non-polar, solvent extractions, or supercritical CO2 extractions and recrystallizations in order to isolate the carotenoids from the other components of the mass. At the end of such purification processes, the carotenoids, as expected, occur in crystalline form.
In the process of the present invention the micelles, microemulsions and nanoemulsions of oxycarotenoid derivatives are obtained during the reaction of such oxycarotenoids with short chain organic acids, such as acetic or propionic acid. Such microscopic emulsions occur during the course of such reaction under controlled conditions and further processing. The carotenoid derivatives obtained by this process have melting points that are lower as compared to the melting points of the free carotenoids. Surprisingly, the carotenoid derivatives crystals under certain conditions of temperature, time, and in the presence of lipids, emulsifiers, and moisture, form stable micelles occluded in the lipid matrix and remain as such at normal conditions of temperature and pressure. Such lipid carotenoid micelles contain melt down oxycarotenoid derivatives, and are non-crystalline.
Furthermore, the present invention is related to a method for improving the absorption and bioavailability of carotenoids by humans and animals by providing a microscopic physical state of xanthophylls or oxycarotenoids derivative forms that are readily incorporated in the digestive system as micelles in the diet of humans and animals.